Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand, is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
______________________________________ ligand + labeled ligand + receptor .fwdarw. ligand-receptor + labeled ligand-receptor. ______________________________________
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for drug derivatives, such as phenobarbital and phenytoin, in serum can be based on competition of an enzyme labeled analogue of such drugs with the drug in the patient serum for immobilized antibody binding sites.
Specific requirements for the labeled drug hapten analogues(hereafter sometimes LDH) include: 1) at least 65% of the LDH can be bound by excess immobilized antibody; 2) affinity of the LDH for immobilized antibody is such that competition of a fixed amount of LDH with the drug occurs in a therapeutically relevant drug concentration range; and 3) stability of the LDH against hydrolysis of its enzyme label under storage conditions.
Requirements imposed on the drug hapten analogue include: 1) accessibility of the analogue to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the labeled analogue by the antibody to the drug; and 3) sufficient reactivity of the analogue with the enzyme label, either directly or following activation of the enzyme or the analogue, under conditions that do not adversely affect enzyme activity.
Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labels coupled to phenobarbital and phenytoin hapten analogues disclosed in U.S. Pat. No. 4,752,568 gave adequate enzyme labeled analogues for conducting effective competitive immunoassays in the desired format.
The problem is that the labeled phenobarbital and phenytoin analogues disclosed in the above patent were unsatisfactory for conducting competitive immunoassays when the enzyme horseradish peroxidase (HRP) was used as the label. The coupling reactions between such analogues and HRP were both slow and incomplete. Moreover phenobarbital and phenytoin HRP labels were bound very weakly so that much higher concentrations of label or antibody binding sites would be required to give a readable signal.